Process for the preparation of an organ-specific substance labeled with technetium-99m

ABSTRACT

A process for the preparation of an organ-specific substance labeled with technetium-99m 
     The invention relates to a process for the preparation of an organ-specific substance labeled with technetium-99m, in which no unwanted Tc-99m compounds are bound to the antibody. This is achieved by using the complexing agent required for the reducing agent in an amount which is stoichiometric relative to the reducing agent.

This is a Rule 60 continuation of application Ser. No. 08/363,577 filedDec. 22, 1994, now abandoned, which is a continuation of applicationSer. No. 08,217,764 filed Mar. 25, 1994, now abandoned, which is acontinuation of application Ser. No. 08,035,521 filed Mar. 12, 1993, nowabandoned, which is a continuation of application Ser. No. 07,829,261filed Feb. 3, 1992, now abandoned.

The invention relates to a process for the preparation of anorgan-specific substance which is labeled with technetium-99m and whichhas been pretreated or coupled to a complexing agent for technetium-99m,where the organ-specific substance is mixed with pertechnetate-99m and acomplex-stabilized reducing agent.

Proteins have been used successfully for many years in medical diagnosiswith radioactive nuclides. Thus, for example, investigations on theheart can be carried out with human serum albumin (HSA) labeled withtechnetium-99m or other suitable nuclides.

Recently, immunoglobulins have become of prime importance, andmonoclonal antibodies in particular are employed for diagnosingmalignant lesions. Monoclonal antibodies have also proven useful inother areas of nuclear medicine diagnosis, for example in the locationof foci of inflammation.

Initially, the antibodies were labeled with various iodine isotopes(iodine-123 or iodine-131) or indium-111. However, clinical trialsshowed that the use of the nuclide technetium-99m (Tc-99m), which has avery much shorter life, is likewise possible. This nuclide occupies anoutstanding position in nuclear medicine because it has very favorablenuclear physical properties. In addition, the Mo-99/Tc-99m generatormakes it available virtually at any time and at any location.

This is why a number of processes in which proteins are labeled withtechnetium-99m have been described. These can be divided into two largegroups.

To be assigned to one group are all the processes which initially couplecomplexing agents of a wide variety of types to the antibody and, viathese, stably bind the technetium-99m. However, this method has thedisadvantage that the labeling yields are inadequately high andtherefore purification steps are necessary for preparing the productready for administration.

The method in which the technetium-99m is directly bound to the antibodybelongs in the second group. Reactive groups are generated in theantibody for this purpose. These are generally SH groups which aregenerated by reduction of disulfide bridges with suitable reducingagents (for example 2-mercaptoethanol, 3-mercapto-1,2propanediol,cysteine etc.). Proteins labeled in this way can be injected directlywithout special purification steps. The following processes from thisgroup should be specifically picked out.

B. A. Rhodes describes the labeling of an anti-hCG antibody with Tc-99m(U.S. Pat. No. 4,472,371). The antibody is in this case treated with anexcess of tin(II) ions which remains in the product. Tin(II) ions bringabout the reduction of disulfide bridges to SH groups in the antibodyand, on the other hand, also the reduction of pertechnetate-99m toTc(IV)-99m. Tc-99m in this form is able to be bound to the antibody.Because of the large excess of Sn(II) ions, not only does Tc-99m occurbound to the antibody but there is also formation of Tc compounds whichmust be removed (cf. U.S. Pat. No. 4,472,371: Example II). However, thisis a serious disadvantage in terms of the conditions which must bemaintained in the laboratory.

EP-A-0,271,806 describes a labeling method in which tin(II) ions arestored in complexed form separate from the antibody. Only shortly beforethe labeling are the two components mixed. The labeling unit thereforecomprises two bottles. This is a disadvantage for the user because hehas to manipulate two bottles which must not be mixed up.

The complex-stabilized Sn(II) ions are used as reducing agent in thiscase too. The complexing agent is employed in excess relative to Sn(II)ions, i.e. there is formation of complexes not only with Sn(II) ions butalso with Tc-99m.

In other prior art publications, the Tc-99m complexes which are formed(with the complexing agent of Sn(II)) are deliberately generated inorder to transfer Tc-99m to the antibody by means of the complex whichis formed. The process is called transcomplexation:

According to EP-A-0,237,150, a Tc-99m tartrate or glucoheptonate isemployed to label an antibody with Tc-99m.

Tc-99m complexes with sucrose, glucoheptonate, tartrate and arabonateare used in WO 88/07382 for the same purpose. Particular importance isin fact attached to the stability of these complexes.

Tc-99m tartrate is used for transcomplexation in WO 89/07456 too. Aprocess is described therein in which the antibody is initially bound toa complexing agent.

The said processes in which Tc-99m is transferred to the antibody bytranscomplexation have the disadvantage, due to the system, that thefinished product contains unwanted Tc-99m compounds as a consequence ofan uncontrollable reaction.

It was therefore an object of the present invention to provide a processfor the preparation of an organ-specific substance labeled withtechnetium-99m, in which no unwanted Tc-99m compounds are bound to theantibody.

The object has been achieved by providing a process of the typedescribed in the introduction, which comprises using the complexingagent which is required for the reducing agent in an amount which isstoichiometric relative to the reducing agent.

It has been surprisingly found--in contrast to previous ideas--that theprocess of transcomplexation is not necessary for transferring Tc-99m tothe antibody.

It was completely surprising to realize that the complexing agent merelyhas the task of keeping Sn(II) ions in solution in a complex, i.e.preventing precipitation as Sn(II) hydroxide. The excess, hitherto used,of complexing agent which initiates the transcomplexation is unnecessaryand, in the final analysis, also has considerable disadvantages: interalia it has been necessary to store Sn(II) ions and antibody in twodifferent vessels.

By contrast, the process according to the invention makes it possiblefor Sn(II) complex and antibody to be stored in one vessel.

Dithionite or Sn(II) ions are preferably used as reducing agent.

By the "stoichiometric" amount of complexing agent is meant the amountwhich is necessary to saturate completely the valencies of the Sn(II)ions and, at the same time, however also to achieve a complex of definedcomposition.

Stoichiometric Sn(II) complexes ought to be stable in a pH range from 3to 11, preferably 4 to 7.

A general statement of the particular stoichiometric ratio of Sn(II)ions to the complexing agent cannot be given because this depends on thecomplexing agent chosen in each case.

Anionic complexes are particularly preferred. Among these in turn thosewith a 4-fold to a 8-fold negative charge. Charge equalization isensured by alkali metal, alkaline earth metal or NH₄ ⁺ ions.

The complexes of the Sn(II) ion with citric acid are very particularlypreferred. The stoichiometric anionic Sn(II)-citric acid complexSn(citrate)₂ !⁴⁻ which forms is known (Gmelin, Handbuch derAnorganischen Chemie, Zinn (Handbook of Inorganic Chemistry, Tin) partC, pages 217-229, Heidelberg 1975).

By contrast, the complex, which is likewise particularly preferred, ofthe Sn(II) ion with 1,1,3,3-propanetetraphosphonic acid has not hithertobeen described. However, it can be obtained by methods known to theperson skilled in the art, in which 1,1,3,3-propanetetraphosphonic acidis reacted with Sn(II) in the molar ratio of 2:1 to one another, andforms a complex whose composition corresponds to this molar ratio (seebelow).

By contrast, a molar ratio below that mentioned would lead to theformation of a sparingly soluble compound in which one molecule ofphosphonic acid binds 4 Sn(II) ions.

Organ-specific substances according to the process according to theinvention are in general carrier substances which have in their moleculeat least one functional group with complexing properties. These groupsare usually atoms or ions which act to donate an electron pair (Lewisbases). One such functional group with complexing property is, forexample, an --SCN, --NH₂, --NHR, --NR₂, --COO, --OH, ═S, --SH, --NOgroup.

Examples of representatives of such substances with functionalcomplexing groups which may be mentioned are: proteins (--NH, --NH₂ orCOO groups), enzymes (--NH₂, --OH, --P═O groups), sugars (--OH groups)or polymers which have side chains with appropriate functional groups.

If the compound to be labeled does not have such a functional group, thesubstance must, before the labeling, be "pretreated" or coupled to asuitable complexing agent.

By "pretreated" are meant within the scope of the invention thosemeasures which lead to the production of a functional group withcomplex-forming properties in the molecule to be labeled. For example,antibodies contain disulfide bridges. However, the two sulfur atomswhich are covalently linked to one another are not in this form able tocomplex technetium-99m. However, reduction of the disulfide bridgeproduces two SH groups which now themselves represent excellentcomplexing ligands for technetium-99m and, moreover, bind the latter ingood yields.

Another possibility for binding technetium-99m to organ-specificsubstances which do not have a functional group with complexingproperties comprises incorporating such a functional group into themolecule or chemically bonding a complexing agent to the molecule.

The process appears particularly interesting for the technetium-99mlabeling of antibodies. Partial reduction of the S--S bonds of theantibody or of an F(ab')₂ antibody fragment can be achieved at roomtemperature by brief exposure to mild reducing agents (pretreatment ofthe organ-specific substance). Particularly suitable reducing agents aremonothiols such as 2-mercaptoethanol or 2-mercaptoethylamine(cysteamine). Obtained in this case are reactive antibody moleculeswhich have neither lost their immunological reactivity nor beenfragmented to smaller fragments. Suitable in principle for the partialreduction of the antibody or the F(ab')₂ antibody fragment are allreducing agents which cleave only some of the S--S bonds even on alengthy exposure time and do not lead to any fragmentation of theantibody component. The time the antibody component is exposed to areducing agent of this type does not need to exceed one hour. Ingeneral, after only 10 to 30 minutes sufficient SH groups have beenproduced for adequate amounts of technetium-99m cations to be bound. Theexcess reducing agent is then removed and the partially reduced antibodyis isolated in a buffered solution (for example 0.02M phosphatesolution, pH 7.2) and lyophilized without delay. It is necessary tosuppress reoxidation of the free thiol groups in the antibody byatmospheric oxygen during this. The lyophilized antibody which, apartfrom the buffer substances, contains no further additives and isblanketed with nitrogen as protective gas can be stored at refrigeratortemperature (-5° to +5° C.) for weeks without alteration; it redissolvessatisfactorily on addition of isotonic sodium chloride solution.

The partially reduced antibody component prepared in this way(pretreated organ-specific substance) can now be labeled smoothly withTc-99m by the process according to the invention when a mixture ofpertechnetate and stoichiometric Sn(II) complex is added to it.

The procedure for the preparation of a diagnostic aid ready for use cannow be such that first the lyophilized antibody component is dissolvedin a technetium-99m-pertechnetate solution and then the reduction andbinding of the technetium to the antibody is brought about by adding asolution of the tin(II) complex.

However, the diagnostic aid can also be prepared by first dissolving theantibody component in the tin(II) complex-containing solution andsubsequently labeling the lyophilized antibody component with technetiumby addition of technetium-99m-pertechnetate solution.

To prepare a diagnostic aid which contains a technetium-99m-labeledorgan-specific substance it is expedient to put together an assay whichcontains the organ-specific substance or the pretreated organ-specificsubstance or the organ-specific substance coupled to a complexing agentfor Tc-99m, where appropriate mixed with a buffer, and thecomplex-stabilized tin(II) salt which is required to reduce thetechnetium on the organ-specific substance. An assay in which thelyophilized, where appropriate pretreated, organ-specific substance ismixed with disodium hydrogen phosphate (pH 7.2) as buffer substance hasproven particularly useful. In this way, after a short reaction time,for example after only 5 minutes, there is obtained virtuallyquantitative technetium-99m-labeling of the substance, which containsless than 1% free pertechnetate and only very small amounts ofTc-99m-labeled tin(II) component as impurities, so that subsequentpurification processes are no longer necessary.

The organ-specific substance prepared by the process according to theinvention ensures--although it is stored in one vessel with the Sn(II)complex--unaltered stability of the lyophilized product. This ensuresrapid, straightforward and satisfactory labeling of the organ-specificsubstance.

The stabilizer present in some labeling kits is also advantageous inantibody labeling because it guarantees an even longer stability of theinjection solution.

The examples which follow are intended to illustrate the invention:

EXAMPLE 1 Preparation of a Stable Complex Between Tin(II) and1,1,3,3-propanetetraphosphonic Acid

0.5240 g (1.0 mmol) of tetrasodium 1,1,3,3-propanetetraphosphonatetetrahydrate (PTP) is dissolved in 100 ml of water. 0.2257 g (1.0 mmol)of tin(II) dichloride dihydrate is dissolved in 100 ml of 0.1Nhydrochloric acid. In a beaker, 2 ml (0.02 mmol) of the PTP solution in1 ml (0.01 mmol) of the tin(II) solution are mixed together and the pHis adjusted to a required value between pH=3 to 11 with 2N sodiumhydroxide solution. A clear solution is obtained.

Mixing of 1 ml (0.01 mmol) of the PTP solution and 1 ml (0.01 mmol) ofthe tin(II) solution immediately results in a precipitate which cannotbe dissolved even by changing the pH in the range from pH=3 to 11.Detectable in the supernatant are no tin(II) ions but still 3equivalents of PTP.

The preparation of the tin(II) citrate complex is described in Example2.

EXAMPLE 2 Preparation of a Tin(II) Complex With Citric Acid

0.12955 g of anhydrous citric acid and 0.07603 g of tin(II) dichloridedihydrate are dissolved in 10 ml of water. The solution is diluted to900 ml with water, the pH is adjusted to pH=6.5 to 7 with 2N sodiumhydroxide solution and then the volume is made up to exactly 1 l withwater. The solution contains 40 μg of Sn(II) per ml and can be useddirectly for the preparation of the antibody products.

The following example shows the preparation of a labeling kit with themonoclonal antibody BW 494/32. This antibody reacts with antigens whichare expressed mainly by mammary or ovarian carcinoma cells.

EXAMPLE 3 Preparation of a Labeling Unit With the Monoclonal Antibody BW494/32

0.5 ml of the solution from Example 1 or Example 2 is added to 1 ml of asolution of the antibody, containing 1 mg of immunoglobulin, which hasbeen treated with 2-mercaptoethanol, 3-mercapto-1,2-propanediol or withanother suitable reducing agent. The solutions are mixed together andthen freeze-dried.

The procedure for labeling with Tc-99m is as follows.

The eluate from a commercially available generator with an activity of500 MBq to 1500 MBq is added to the lyophilizate. This activity can bepresent in a volume of 1 ml to 10 ml. The product is ready for injectionafter a time of 5 min to 10 min has elapsed.

Examination of the radiochemical purity by thin-layer chromatography(ITLC SG/methyl ethyl ketone) or high performance liquid chromatography(gel filtration column (for example Bio Rad TSK 250), 0.1M phosphatebuffer pH 6.8, flow rate: 1 ml/min) showed that between 95% and 99% ofthe added activity was bound to the antibody. This solution remainedsufficiently stable until a time of up to 24 hours had elapsed.

In an animal experiment on nude mice with tumors, storage levels in thetumor between 6 and 7%/g of tumor were achieved with these products.

We claim:
 1. A diagnostic aid prepared by initially dissolving a component which contains an organ-specific substance in a technetium-99m-pertechnetate solution, and then bringing about reduction and direct binding of the technetium to the organ-specific substance by adding a complex-stabilized reducing agent, wherein the complex-stabilized reducing agent is a stoichiometric complex of Sn(II) with either citric acid or 1,1,3,3-propanetetraphosphonic acid in a molar ratio of about 1:2.
 2. A diagnostic aid as claimed in claim 1, wherein 1 to 100 μg of said reducing agent is added per 1 mg of the organ-specific substance for stable labeling of the latter with technetium-99m.
 3. A diagnostic aid as claimed in claim 1 wherein the amount of complex-stabilized reducing agent is 5 to 10 μg, based on Sn(II), per 1 mg of the organ-specific substance for stable labeling of the latter with technetium-99m.
 4. A diagnostic aid prepared by initially dissolving a component which contains an organ-specific substance in a solution of a complex-stabilized reducing agent, and subsequently directly labeling the organ-specific substance with technetium by adding technetium-99m pertechnetate solution, wherein the complex-stabilized reducing agent is a stoichiometric complex of Sn(II) with either citric acid or 1,1,3,3-propanetetraphosphonic acid in a molar ratio of about 1:2.
 5. A process for the preparation of an organ specific substance directly labeled with technetium-99, comprising the steps of:a) choosing an organ specific substance with a functional group with complex-forming properties, or pretreating an organ specific substance to produce, on the organ specific substance, a functional group with complex-forming properties; b) preparing a complex-stabilized reducing agent by combining a complexing agent, selected from the group consisting of citric acid and 1,1,3,3,-propanetetraphosphonic acid, with an Sn(II) reducing agent wherein the complexing agent and reducing agent are present in a molar ratio of about 2:1; and c) mixing the organ-specific substance of step (a) with pertechnetate-99m and the complex stabilized reducing agent of step (b).
 6. The process as claimed in claim 5, wherein the complex of the reducing agent is stable in a pH range from 3 to
 11. 7. The process as claimed in claim 5, wherein the complex is an anionic complex.
 8. The process as claimed in claim 5, wherein said organ-specific substance is a protein.
 9. The process as claimed in claim 5, wherein said organ-specific substance is a monoclonal antibody or its F(ab')₂ fragment.
 10. The process as claimed in claim 9, wherein said antibody or its F(ab')₂ fragment is specific for a tumor-associated antigen.
 11. The process as claimed in claim 5, wherein said organ-specific substance is an enzyme.
 12. The process as claimed in claim 5, wherein said organ-specific substance is a sugar.
 13. The process as claimed in claim 5, wherein said organ-specific substance is a polymer. 